Posttranscriptional Derepression of GADD45α by Genotoxic Stress Ashish Lal, Kotb Abdelmohsen, Rudolf Pullmann, Tomoko Kawai, Stefanie Galban, Xiaoling Yang, Gary Brewer, Myriam Gorospe  Molecular Cell  Volume 22, Issue 1, Pages 117-128 (April 2006) DOI: 10.1016/j.molcel.2006.03.016 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 Increased GADD45α mRNA Stability after MMS Treatment (A and B) The levels of GADD45α mRNA, GAPDH mRNA, and loading control 18S rRNA after MMS treatment of HeLa cells (at the indicated times and concentrations) were monitored by Northern blotting (A) and by RT-qPCR (B). (C) HeLa cells were either left untreated or treated with MMS (100 μg/ml) for 4 hr, whereupon RNA was prepared from nuclear and cytoplasmic fractions and used to monitor GADD45α mRNA levels (and those of loading controls 18S rRNA and GAPDH mRNA) by RT followed by conventional PCR (gel images, depicting 25, 20, and 15 cycles, respectively, for GADD45α, GAPDH, and 18S) and qPCR (graphs). (D) To estimate mRNA half-life, cells were treated with MMS for 2 hr and then with actinomycin D (5 μg/ml) for the times shown. GADD45α and GAPDH mRNA levels were measured by RT-qPCR, normalized to 18S rRNA levels, and plotted on a logarithmic scale to calculate the time required for each mRNA to reach one-half of its initial abundance (50%, dashed line). (E) Western blot analysis of Gadd45α expression (and loading control α-tubulin) in whole-cell lysates (100 μg/lane), after 6 hr of MMS treatment. All graphs represent the means ± standard errors of the means (SEM) from at least three independent experiments. Molecular Cell 2006 22, 117-128DOI: (10.1016/j.molcel.2006.03.016) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 AUF1 and TIAR Bind Endogenous and Biotinylated GADD45α Transcripts (A) Top, the association of endogenous ARE binding proteins (HuR, AUF1, TIAR, TIA-1, and KSRP) to endogenous GADD45α mRNA was assessed by RT-PCR analysis of the RNA obtained after IP of HeLa whole-cell lysates by using either IgG or specific antibodies. Low-level (background) amplification of GAPDH in the IP material served as normalization control. Bottom, representative IP followed by Western blotting (WB) detection of the RBPs tested; abbreviation: H.C., heavy chain. (B) Schematic of GADD45α mRNA depicting the transcripts (5′UTR, CR, and 3′UTR RNAs A, B, and C) used for biotin pull-down analysis. Using HeLa whole-cell lysates, the binding of AUF1 and TIAR to equimolar amounts of biotinylated GADD45α RNA fragments was tested by biotin pull down followed by Western blotting (details in the Supplemental Data). Molecular Cell 2006 22, 117-128DOI: (10.1016/j.molcel.2006.03.016) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 AUF1 Lowers GADD45α mRNA Levels and Dissociates after MMS Treatment (A) Whole-cell lysates were prepared from HeLa cells (untreated or treated with MMS [100 μg/ml, 4 hr]), and the abundance of GADD45α mRNA (and GAPDH mRNA, present at low levels in the IP material and, hence, serving as normalization control) was assessed either by RT-PCR (top) or by RT-qPCR (bottom) after IP with either anti-AUF1 or IgG antibodies. (B) AUF1, α-tubulin (a cytoplasmic protein marker), and hnRNP C1/C2 (a nuclear protein marker) were detected by Western blotting using total, cytoplasmic, and nuclear extracts (20 μg each) prepared from either untreated or MMS-treated cells. (C) HeLa cells were treated as described in (A) and AUF1 detected by IP followed by Western blot analysis; abbreviation: L.C., light chain. (D) After transfection with the vector alone (V) or with plasmids overexpressing specific isoforms of AUF1 (p37, p40, p42, and p45), AUF1 and the loading control α-tubulin were detected by Western blot analysis of whole-cell lysates (top); abbreviation: N.S., nonspecific band. The effect of overexpressing AUF1 isoforms on the levels of mRNAs encoding GADD45α and GAPDH (loading control) was monitored by RT-qPCR analysis of transfected cells. (E) After transfection as described in (D) and MMS treatment (100 μg/ml, 4 hr), changes in the levels of mRNAs encoding GADD45α and p21 (and normalization controls GAPDH and UBC) were assessed by RT-qPCR. Graphs represent the means ± SEM from at least three independent experiments. Molecular Cell 2006 22, 117-128DOI: (10.1016/j.molcel.2006.03.016) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 AUF1 Downregulation Elevates GADD45α Expression Levels (A and B) After transfection with either a control plasmid (V) or a plasmid expressing an shRNA capable of downregulating AUF1, the abundance of AUF1, loading control α-tubulin, and other RBPs were assessed by Western blotting in whole-cell lysates (A); the changes in levels of GADD45α mRNA and control mRNAs encoding p21 (regulated by AUF1), UBC, and GAPDH (not regulated by AUF1) were monitored by RT-qPCR and shown as the means ± SEM from three independent experiments (B). (C) mRNA half-lives were measured as described for Figure 1D (data are the average from two independent experiments, each showing similar results). Molecular Cell 2006 22, 117-128DOI: (10.1016/j.molcel.2006.03.016) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 MMS Triggers the Dissociation of GADD45α mRNA from TIAR and Increases the Polysomal Presence of GADD45α mRNA (A) The polysome-associated mRNAs encoding GADD45α, p21 (regulated by MMS), UBC, and GAPDH (not regulated by MMS) were measured by RT-qPCR in HeLa cells that were either left untreated or treated with MMS (100 μg/ml, 4 hr). (B) RNA was extracted from each fraction and GADD45α and GAPDH mRNA levels tested by RT-qPCR (see also Figure S2). Data are represented as a the percentage of the total mRNA detected in each fraction of each treatment group; inset graph, relative mRNA abundance in each fraction of each group (GADD45α mRNA was higher overall in MMS-treated cells). (C) Whole-cell lysates were prepared from either untreated or MMS-treated cells, and the abundance of GADD45α mRNA (and GAPDH mRNA, present at low levels in the IP material and thus serving as normalization control) was assessed by RT-PCR (top) and by RT-qPCR (bottom) after IP with either anti-TIAR or IgG antibodies. (D) TIAR, α-tubulin (a cytoplasmic protein marker), and hnRNP C1/C2 (a nuclear protein marker) were detected by Western blotting using total, cytoplasmic and nuclear extracts (20 μg each) prepared from cells that were treated as described in (C). (E) Cells were treated as described in (C), and TIAR was detected by IP followed by Western blotting. (F) HeLa cells were transfected with a control siRNA (Ctrl. siRNA) or siRNA targeting TIAR (TIAR siRNA) and the silencing of TIAR monitored by Western blotting; α-tubulin served as the loading control. (G) Changes in the levels of polysome-associated GADD45α mRNA (or control GAPDH and UBC mRNAs) after TIAR silencing were determined by performing RT-qPCR analysis of polysomal RNA isolated from cells that were transfected as in (F). (H) Left, anti-AUF1 and control IgG IPs were performed after silencing TIAR as described in (F); right, anti-TIAR and IgG IPs were performed after silencing AUF1 as described in Figure 4. The levels of GADD45α and GAPDH mRNAs were subsequently measured in the IP samples. Data represent mRNA levels in anti-AUF1 IP relative to IgG IP (left) and mRNA levels in anti-TIAR IP relative to IgG IP (right). Graphs depict the means ± SEM from at least three independent experiments. Molecular Cell 2006 22, 117-128DOI: (10.1016/j.molcel.2006.03.016) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 6 The 3′UTR of GADD45α mRNA Bears MMS-Responsive Regulatory Elements (A) Schematic of plasmids used in transfections to express either EGFP mRNA (pEGFP) or a chimeric RNA comprising EGFP and the GADD45α 3′UTR [EGFP-G45(3′)]. The EGFP coding region amplified by RT-qPCR is indicated. (B) HeLa cells were transfected with the constructs described in (A), and 48 hr later, the levels of EGFP expressed from pEGFP and pEGFP-G45(3′) expression constructs were tested by Western blotting (left) and the levels of each transcript were measured by RT-qPCR (right). EGFP from the EGFP mRNA is 3 kDa larger than that expressed from the EGFP-G45(3′) mRNA due to the cloning strategy employed (Supplemental Data); α-tubulin served as a loading control. (C) Cells transfected as described in (B) were treated with MMS (100 μg/ml) for the indicated time intervals, and RT-qPCR was performed to monitor changes in EGFP or EGFP-G45(3′) mRNA levels; GAPDH mRNA served as a normalization control. In (B) and (C), graphs represent the means ± SEM from at least three independent experiments. (D) After transfection and treatment as described in (C), Western blot analysis was performed to assess changes in EGFP protein expressed from either EGFP or EGFP-G45(3′) mRNAs (EGFP signals were measured by densitometry and shown as the percentage of EGFP in untreated cells); hnRNP C1/C2 levels were monitored to ensure equal loading. Molecular Cell 2006 22, 117-128DOI: (10.1016/j.molcel.2006.03.016) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 7 Downregulation of AUF1 or TIAR Relieves the GADD45 3′UTR-Mediated Suppression (A–F) HeLa cells were cotransfected with vectors pEGFP or pEGFP-G45(3′), along with either a control plasmid (V) or a plasmid expressing an AUF1-directed shRNA (AUF1 shRNA). Forty-eight hours later, the levels of EGFP expressed from each vector were monitored by Western blotting (A) and the corresponding mRNAs by RT-qPCR (B); in the latter analysis, transcript levels in MMS-treated and untreated cells were compared and GAPDH mRNA levels quantified to assess sample differences. Cells were cotransfected with vectors pEGFP or pEGFP-G45(3′), along with either control (Ctrl.) or HuR-targeting (HuR) siRNAs. Forty-eight hours later, the levels of EGFP expressed from each vector were monitored by Western blot analysis (C) and the corresponding mRNAs by RT-qPCR (D); in the latter analysis, the levels of mRNAs encoding GAPDH (loading control), cyclin D1 (a target of HuR and hence a positive control), and endogenous GADD45α were also measured. HeLa cells were cotransfected with vectors pEGFP or pEGFP-G45(3′), along with either control (Ctrl.) or TIAR-targeting (TIAR) siRNAs. EGFP expressed from each vector was monitored by Western blot analysis (E) and the corresponding mRNAs by RT-qPCR (F) 48 hr later. In (B), (D), and (F), graphs represent the means ± SEM from at least three independent experiments. (G) Changes in nascent EGFP (35S-EGFP), translated from either pEGFP or pEGFP-G45(3′), were assessed by incubating transfected cells with L-[35S]methionine and L-[35S]cysteine for 20 min. After IP using either IgG or anti-EGFP antibodies (or anti-α-tubulin to monitor differences in sample processing), proteins were resolved by SDS-PAGE and transferred onto membranes for visualization of radiolabeled signals (percentage signal intensities of nascent EGFP are shown). In (A), (C), (E), and (G), α-tubulin levels served to monitor equal loading. Molecular Cell 2006 22, 117-128DOI: (10.1016/j.molcel.2006.03.016) Copyright © 2006 Elsevier Inc. Terms and Conditions